Angiogenesis is a process in which new blood vessels are generated from existing blood vessels, and it is known that angiogenesis is closely involved in an onset and development of diseases such as malignant (solid) tumor, diabetic retinopathy, or retinal angiogenesis, inflammatory diseases (rheumatism, etc.). For example, in order for solid tumors to grow, it is necessary to secure a supply route of nutrition and oxygen and an elimination route of wastes by angiogenesis. Angiogenesis plays an important role for tumor metastasis, an issue special concern for cancer treatment, since angiogenesis secures the blood supply. As for diabetic retinopathy, angiogenesis itself is a pathological condition, and patients lose their eyesight if left untreated. Therefore, it is considered that inhibition of angiogenesis leads to prevention/treatment of diseases, and preventing/treating agents for angiogenesis are currently explored.
Since angiogenesis promotes various pathological conditions mentioned above, inhibition of angiogenesis is expected to be beneficial in prevention/treatment of such conditions. For the purpose of prevention or treatment of diseases associated with angiogenesis, studies in search for angiogenesis-inhibiting substances have been performed. As a result, many angiogenesis-inhibiting substances have been identified, and for some of them, clinical usefulness is now under investigation.
For instance, angiogenesis inhibitors such as endostatin and angiostatin were once known to be the most potent agents for tumor dormancy therapy. They were expected to serve as ideal anticancer drugs with least adverse reactions because their systemic therapy regressed solid tumors in experimental animals remarkably (Cell, 88, 277-285, 1997) without any acquired resistance as experimental tumors do not develop resistance to multiple cycles of therapy unlike conventional anticancer drugs (Nature, 390, 404-407, 4997). However, in clinical practice, synthesis of an effective dosage of these high molecular proteins to elicit antitumor effect is difficult and costly and, consequently, business circles have already abandoned clinical applications of angiostatin, whose molecular weight is about 50000.
Endostatin, with lower molecular weight (about 20000), attracted attention and its clinical applications have started in terminal malignant tumor patients in U.S.A. However, its precise mechanisms of action as well as its receptors had been unknown.
Endostatin inhibits the proliferation of endothelial cells and induces apoptosis under reduced serum culture condition (J. Biol. Chem., 274, 11721-11726, 1999), but since the effect was limited, and it was difficult to ascribe the potent effect to regress primary and metastatic cancers. Tumor cells attain accelerated proliferative characteristics not only by genomic mutation and deregulated gene expression but also by vigorously secreting many growth- and angiogenesis-promoting factors in an autocrine/paracrine fashion; and further, newly generated blood vessels supply abundant blood flow. In order for endostatin to inhibit tumor angiogenesis under such circumstances as currently reported, potent intracellular signals specifically acting on endothelial cells must be induced. These mechanisms have been unknown for long time.
On the other hand, in 1957, P. Sensi et al. of Lepetit Research Laboratories in Italy separated Streptomyces mediterranei (later, classified into Nocardio mediterranei) from soil collected at the coast of the Mediterranean, and obtained rifamycin, an antibiotic showing antibacterial activity to acid-fast bacteria and Gram-positive bacteria from the culture liquid thereof. Rifamycin in the culture liquid is a mixture comprising rifamycins A, B, C, D, E, etc., and rifamycin O is an oxidized type of rifamycin B. Rifamycin B and rifamycin O are induced into rifamycin S, and rifamycin S is reduced to rifamycin SV by ascorbic acid. 3-formyl rifamycin is made by 3-formylation of rifamycin. Rifampicin is induced from a substance made by 3-formylation of rifamycin SV. Rifamycin is collectively called as ansamycin antibiotics because it has an aromatic ring system to which an aliphatic bridge called ansa ring is connected.
In addition, the above-mentioned rifampicin is an ansamycin antibiotic developed from a collaboration of Ciba-Geigy (Switzerland) and Lepetit (Italy), and is induced from a substance made by 3-formylation of rifamycin SV. In other words, rifampicin is an ansamycin semisynthetic antibiotic having a structure of 3-{[(4-methyl-1-piperazinyl)imino]methyl}rifamycin, and is a substance which has excellent antituberculosis activity and has been used widely as an anti-tuberculous drug. Rifampicin has antibacterial activity not only to Gram-positive bacteria but also to Gram-negative bacillus, and has been used for brucellosis, chlamydia infection, and infection of Gram-positive bacteria such as staphylococcus as well as tuberculosis.
Rifampicin is synthesized by reacting 3-formyl rifamycin SV with 1-amino-4-methylpiperazine in tetrahydrofran, and many synthetic methods including industrial synthetic methods are disclosed (Japanese Patent Publication Nos. 42-26800, 47-23303, 53-39400, 57-40155, 62-41671, 62-41672, and 62-41673).
The object of the present invention is to provide a novel angiogenesis inhibitor which is safe and highly practical, more particularly, a novel angiogenesis inhibitor which is effective, safe and highly practical for inhibition of angiogenesis in various diseases such as malignant tumor, diabetic retinopathy, retinal angiogenesis and inflammatory diseases. In addition, the present invention provides a method for screening a novel angiogenesis-inhibiting substance which serves as an active ingredient of an angiogenesis inhibitor.
As a result of intensive search to attain the objective mentioned above, the present inventors have found that rifampicin, which has excellent antituberculous and antibacterial activity to both Gram-positive and negative bacteria and widely used to treat brucellosis, chlamydia infection, and staphylococcus infection as well as tuberculosis, has excellent angiogenesis-inhibiting activity. This led to the completion of the present invention. The present invention, further confirmed that ansamycin antibiotics such as rifamycin SV or 3-formyl rifamycin have angiogenesis-inhibiting activity as well.
In the present invention, the finding that ansamycin antibiotics such as rifampicin, etc., have excellent angiogenesis-inhibiting activity has its origin in the elucidation of endostatin-induced molecular signals by the present inventors. Recently, the present inventors have found a molecular mechanism involved in inhibition of angiogenesis by endostatin (FASEB Journal 15, 1044-1053, 2001). Administration of endostatin at concentrations showing tumor regression in experimental animals markedly inhibited various immediately early response genes and apoptosis/cell-cycle/migration-associated genes expressed in cultured vascular endothelial cells under supplementation with serum, growth factors and angiogenesis factors.
As a result of down-regulation of a variety of gene expression, endostatin causes marginal endothelial cell proliferation, but marked inhibition of endothelial cell migration. The molecular responses, which are potent and wide spectrum of gene down-regulation by endostatin, are designated as “angiogenesis-inhibiting signals” by a present inventor. By quantifying mRNA levels using real-time quantitative PCR, it becomes possible to rapidly identify substances showing potent signals similar to endostatin among many reagents, and to examine whether they exert potent inhibition of endothelial cell migration/proliferation.
Conventional process of identifying novel angiogenesis-inhibiting factors required repetition of protein purification by extracting and fractionating tumor regressive activity released by tumor themselves from a large amount of body fluid and/or supernatant of cell cultures. The entire process was time-consuming and it took long before gene cloning. Further, it was difficult to synthesize a sufficient dosage of large molecular weight angiogenesis-inhibiting factors, endostatin and angiostatin.
By the method for screening angiogenesis-inhibiting substances which detects “angiogenesis-inhibiting signals” constructed by the present inventors, it becomes possible to detect factors exerting endostatin-type signals among many substances, and to select a novel angiogenesis-inhibiting factor by examining tumor regression activity and angiogenesis-inhibiting activity. This screening method greatly reduces conventional processes, and further, allows to estimate dosages of peptides/proteins/drugs required to induce the effect comparable to that of endostatin in advance.
The method enables to find novel factors clinically applicable to a therapy targeting new blood vessel formation in tumors. Further, when angiogenesis-inhibiting signals by endostatin are confirmed to correlate with tumor regression activity, in vitro activity of many synthetic peptides and compounds efficiently screened can be utilized. A test substance can be added to the supernatant of cultured vascular endothelial cells to detect angiogenesis-inhibiting signals, and, it becomes possible to select efficiently novel angiogenesis inhibitors from many substances. Peptides/proteins with smaller molecular weight and with structures applicable easily clinically can be preferentially selected, while potential side effects may be predicted for ingredients of drugs and food at effective concentrations to induce antiangiogenesis, in advance at the stage of screening.
In the present invention using the above-mentioned screening method, ansamycin antibiotics such as rifampicin have been found to induce strong angiogenesis-inhibiting activity among many candidate substances. Since ansamycins such as rifampicin have been widely used as antibiotics, their safety is well known and method for producing and administering them have been established. Therefore, it is expected that they can be used as highly practical angiogenesis inhibitors.